Autoinducer molecule

ABSTRACT

Autoinducer molecules, e.g., N-(3-oxododecanoyl)homoserine lactone, for  Pseudomonas aeruginosa  are described. The molecules regulate gene expression in the bacterium. Therapeutic compositions and therapeutic methods involving analogs and/or inhibitors of the autoinducer molecules also are described. The molecules are useful for treating or preventing infection by  Pseudomonas aeruginosa.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/541,873 filed on Nov. 19, 2001, which is a Continued ProsecutionApplication of Ser. No. 09/541,873 filed on Apr. 3, 2000, which is aContinuation Application of Ser. No. 08/456,864 filed on Mar. 17, 1999,which in turn is a Continued Prosecution Application of Ser. No.08/456,864 filed Jun. 1, 1995, which in turn is a Divisional Applicationof Ser. No. 08/104,487 filed Aug. 9, 1993.

GOVERNMENT SUPPORT

This research was supported by grants and fellowships from the CysticFibrosis Foundation, Office of Naval Research (N00014-80-6570), NationalInstitute of Allergy and Infectious Diseases (33713), and NationalScience Foundation (DIR9017262).

BACKGROUND

The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistichuman pathogen that causes infections in immunocompromised hosts, andcolonizes the lungs of individuals with cystic fibrosis (Hoiby, N.(1974) Acta Patholgica Microbiol. Scand. Sect. B. 82, 551-558; Reynolds,H. Y., Levine, A. S., Wood, R. E., Zierdt, C. H., Dale, D. C. andPennington, J. L. (1975) Ann. Intern. Med. 82, 819-832). This bacteriumproduces a number of extracellular virulence factors including exotoxinA, which is encoded by the toxA gene (Iglewski, B. H. and Kabat, D.(1975) Proc. Natl. Acad. Sci. USA. 72, 22842288; Iglewski, B. H.,Sadoff, J. C., Bjorn, M. J., and Maxwell, E. S. (1978) Proc. Natl. Acad.Sci. USA. 75, 3211-3215), two elastolytic proteases, encoded by the lasAand lasB genes, and an alkaline protease encoded by the aprA gene(Morihara, K. and Homma, J. Y. (1985) in Bacterial Enzymes andVirulence, ed. Holder, I. A. (CRC Press, Boca Raton, Fla.) pp. 41-79;Bever, R. A. and Iglewski, B. H. (1988) J. Bacteriol. 170, 4309-4313;Kessler, E. and Saffrin, M. (1988) J. Bacteriol. 170, 5241-5247).

Autoinducer molecules are capable of regulating the gene expression ofcertain microorganisms. Bycroft et al. (WO92/18614) describe a class ofautoinducer molecules which includes N-(β-ketocaproyl) L-homoserinelactone and N-(β-hydroxybutyryl) homoserine lactone. All of theexemplified autoinducer molecules of Bycroft et al. contain C₁-C₇ sidechains. However, autoinducer molecules with side chains of greaterlength or cyclic side chains are not exemplified by Bycroft et al.

Bycroft et al. state that Pseudomonas aeruginosa is affected byN-(β-ketocaproyl) homoserine lactone. As recently as 1993, researchershave believed that N-(β-ketocaproyl) homoserine lactone is theautoinducer molecule of P. aeruginosa. (Stewart, G. S. A. B. and P.Williams (1993) ASM News, 59, 241-46)

SUMMARY OF THE INVENTION

The present invention is based, in least in part, on the discovery thatthe autoinducer molecule for Pseudomonas aeruginosa isN-(3-oxododecanoyl)homoserine lactone and notN-(β-ketocaproyl)homoserine lactone as previously believed. Upon thediscovery of this novel autoinducer molecule, it was realized thatautoinducer molecule(s) containing a fatty moiety or a moiety having atleast seven members in the R moiety of the formula set forth below:

are involved in the regulation of gene expression. In the above formula,n is 2 or 3; Y is O, S, or NH; X is O, S, or NH; and R is a fattyhydrocarbon or acyl moiety that may be substituted or a moiety having atleast seven members containing a ring structure that may be substituted.The present invention further pertains to autinducer molecules of thefollowing formula:

wherein X and Y are as defined above and Z₁ and Z₂ are independentlyselected from the group consisting of hydrogen, ═O, ═S, and ═NH.

The present invention also pertains to analogs of the autoinducermolecule that affect the activity of the LasR protein, e.g., inhibit theautoinducer activity or synergistically enhance the autoinduceractivity. The present invention even further includes inhibitors of theautoinducer activity of N-(3-oxododecanoyl)homoserine lactone. Thepresent invention also pertains to methods of selecting such inhibitorsand analogs. These methods involve the contact of the autoinducermolecule with the suspected inhibitor or synergist followed by themeasuring of the ability of the treated autoinducer molecule to performits intended function. From these steps, it is determined whether thesuspected inhibitor or synergist inhibits or enhances the ability of theautoinducer molecule to stimulate the activity of the selected gene.

The present invention also pertains to therapeutic compositionscomprising an agent having the ability to inhibit the activity of theLasR protein of Pseudomonas aeruginosa and/or inhibit the autoinduceractivity of N-(3-oxododecanoyl)homoserine lactone and a pharmaceuticallyacceptable carrier. The agent can be the analogs or inhibitors asdescribed above and in further detail below.

The present invention even further pertains to a method of inhibitingthe infectivity of Pseudomonas aeruginosa and a method of treating animmunocompromised individual infected with Pseudomonas aeruginosa. Bothof these methods involve the administration to an individual of atherapeutically effective amount of the agents and/or therapeuticcompositions described above that inhibit the activity of the LasRprotein and/or inhibit the autoinducer activity ofN-(3-oxododecanoyl)homoserine lactone. An example of animmunocompromised individual in an individual afflicted with cysticfibrosis.

The present invention further pertains to a culture medium containing asan added compound an autoinducer molecule as described and methods ofcontrolling the expression of a gene in bacteria or cells using thedescribed autoinducer molecules and/or analogs or inhibitors thereof.

The present invention also pertains to analogs of the autoinducermolecule that inhibit the induction of virulence factors by theautoinducer molecule or LasR. The virulence factors include exotoxin A,elastolytic proteases, and an alkaline protease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the dose-response curve for P. aeruginosa autoinducer inethyl acetate extracts of bacterial culture fluid. Culture fluidextracts were of P. aeruginosa PA01 (•), P. aeruginosa PA01-R1 (◯), E.coli with pLasI-1 (▪), and E. coli without pLasI-1 (□). As defined inMaterials and Methods, a unit of autoinducer activity is that amountrequired for half-maximal activation of the lasB promoter in E. coli(pKDT17).

FIG. 2 shows the HPLC analysis of autoinducer extracts. Extractedautoinducer was from an E. coli (pLasI-1) culture medium (◯), and from aP. aeruginosa PA01 culture medium (•). Methanol-gradient HPLC was asdescribed in Materials and Methods. Each fraction was 2 ml. The dashedline indicates the methanol concentration. The percent of activityrecovered in the major peak for either bacterium was >75%. The whitetriangle indicates where N-(3-oxohexanoyl)homoserine lactone elutes, andthe black triangle indicates where N-(3-oxo-octanoyl)homoserine lactoneelutes.

FIG. 3 shows the dose-response curve for activity of syntheticN-(3-oxododecanoyl)homoserine lactone in the P. aeruginosa autoinducerbioassay.

FIG. 4 shows the known autoinducer structures. From the top,N-(3-hydroxybutanoyl)homoserine lactone, the inducer of luminescence inVibrio harveyi; N-(3-oxohexanoyl)homoserine lactone, the inducer of V.fischeri luminescence; N-(3-oxooctanoyl)homoserine lactone, the inducerof conjugal transfer genes in Agrobacterium tumefaciens; andN-(3-oxododecanoyl)homoserine lactone, the P. aeruginosa autoinducer ofthe present invention.

DETAILED DESCRIPTION

The present invention pertains to autoinducer molecules of the formula:

wherein n is 2 or 3; Y is O, S, or NH; X is O, S, or NH; and R is afatty hydrocarbon or acyl moiety that may be substituted or a moietyhaving at least seven members containing a ring structure that may besubstituted. The autoinducer molecule regulates the activity of the LasRprotein of Pseudomonas aeruginosa. In addition, the present inventionpertains to optically active isomers of the autoinducer molecule. Theautoinducer molecule can be purified from the native source usingconventional techniques or can be derived synthetically by chemicalmeans. Included in the invention are optically active isomers of theclaimed autoinducer molecule as well as analogs of the claimedautoinducer molecule.

The language “autoinducer molecule” is intended to include a moleculeinvolved in the regulation of gene expression, e.g., it may increase ordecrease gene expression, of a microorganism. Typically, autoinducermolecules are produced by microorganisms, such as bacteria, duringmetabolism. The autoinducer molecules then regulate gene expression, forexample, by combining with a transcriptional activator protein.

The language “fatty hydrocarbon or acyl moiety” is intended to include along straight or branched chain moiety having seven or more carbonatoms. For example, the fatty acyl moiety can be of the followingformula:

wherein n is 4 or more. The preferred fatty acyl moieties include C₇-C₁₄acyl moieties, more preferred are C₁₀-C₁₄ acyl moieties, and mostpreferred is the C₁₂ acyl moiety. Fatty hydrocarbon or acyl moietiesinclude saturated and unsaturated moieties as well as substitutedmoieties, for example, by substituting a S for an O. Examples of fattyhydrocarbon moieties include fatty alkyl, fatty alkenyl, and fattyalkynyl moieties.

The language “ring structure” is intended to include arrangements ofatoms which form one or more rings. The ring structures can containheterocyclic ring(s), e.g., oxygen, sulfur, or nitrogen containing, orcan contain carbocyclic ring(s). The ring structure further can be afused ring system. Examples of ring structures include 5 to 7 memberedheterocyclic rings, napthyl, and phenyl. The ring structures further canbe substituted with groups that do not effect the molecule's ability toperform its intended function as described above.

The substituents on the “R” moiety are substituents which do notdetrimentally effect the molecule's ability to perform its intendedautoinducer function. Examples of such substituents include hydrocarbongroups, e.g., lower alkyl, alkenyl and alkynyl groups, keto groups andhalogen containing substituents. Examples of such substituents includebutyl, propyl, methyl, butenyl, propenyl and butynyl groups.

The language “able to regulate the activity” is intended to include theactivation of or an act to increase the operation of another molecule,e.g., the LasR protein.

The language “LasR protein of Pseudomonas aeruginosa” is intended toinclude the transcriptional activator proteins of the bacteriaPseudomonas aeruginosa. LasR proteins of P. aeruginosa include proteinsencoded by the lasR gene of P. aeruginosa. The LasR proteins are globalregulators of genes involved in the virulence of P. aeruginosa.

The language “isomer” is intended to include molecules having the samemolecular formula as the autoinducer molecule but possessing differentchemical and physical properties due to a different arrangement of theatoms in the molecule. Isomers include both optical isomers andstructural isomers.

The language “optically active” is intended to include molecules thathave the ability to rotate a plane of polarized light. An opticallyactive isomer includes the L-isomer and the D-isomer of the claimedautoinducer molecule. The L-isomer of N-(3-oxododecanoyl)homoserinelactone is the active form. The D-isomer shows a small amount ofactivity and can inhibit the ability of the L-isomer to activate theLasR protein by attaching to the autoinducer binding domain of the LasRprotein.

The language “purified from the native source” is intended to include anautoinducer molecule of the above formula that has been manufactured byan organism. “Purified from the native source” includes isolating theautoinducer molecule from the culture media or cytoplasm of bacteriasuch as Pseudomonas aeruginosa using conventional techniques.

The language “synthesized by chemical means” is intended to includeautoinducer molecules of the claimed formula that have been madeartificially outside of an organism. The invention includes the claimedautoinducer made by a scientist in a laboratory from chemical precursorsusing standard chemical synthesis techniques. For example, the claimedautoinducer molecules can be synthesized using the protocol of Eberhardet al. (Eberhard, A., Burlingame, A. L., Eberhard, C., Kenyon, G. L.,Nealson, K. H. and Oppenheimer, N.J. (1981) Biochemistry 20, 2444-2449)from commercially available precursors. The starting materials can bemodified to produce the desired end product.

The present invention further pertains to autinducer molecules of thefollowing formula:

wherein X and Y are as defined above and Z₁ and Z₂ are independentlyselected from the group consisting of hydrogen, ÍO, ÍS, and ÍNH.

The preferred autoinducer molecule of the present invention is of theformula:

This autoinducer molecule is a novel chemical compound which is at leastpart of the present invention. The new chemical compound has utility asan autoinducer and also may have other utilities. As an autoinducer, itcan regulate the activity of the transcriptional protein of Pseudomonasaeruginosa, LasR. The chemical name of the autoinducer molecule isN-(3-oxododecanoyl)homoserine lactone. The autoinducer molecule can bepurified from the native source or can be derived synthetically bychemical means. Included in the invention are optically active isomersof the claimed autoinducer molecule as well as analogs of the claimedautoinducer molecule.

The language “analog” is intended to include molecules that arestructurally similar but not identical to the claimed autoinducermolecule N-(3-oxododecanoyl)homoserine lactone. For example, the lengthof the fatty acyl moiety can be varied producing an analog or one of theketo groups can be removed from the fatty acyl moiety. Analogs includeautoinducer molecules that are structurally similar to the claimedautoinducer molecule but can inhibit rather than stimulate the activityof the LasR protein or analogs which act synergistically to enhance theability of the claimed autoinducer to increase the activity of the LasRprotein. One of ordinary skill in the art would be able to selectanalogs which are useful within the present invention using theselection methods described below.

The present invention also pertains to methods of selecting inhibitorsor synergists of the autoinducer molecule, N-(3-oxododecanoyl)homoserinelactone. The method comprises contacting the autoinducer molecule with asuspected inhibitor or synergist, measuring the ability of the treatedautoinducer molecule to stimulate the activity of a selected gene thendetermining whether the suspected inhibitor or synergist represses orenhances the activity of the autoinducer molecule. Actual inhibitors andsynergists of the autoinducer molecule are then selected. For example, asuspected inhibitor can be mixed with N-(3-oxododecanoyl)homoserinelactone and the mixture then combined with E. coli MG4 which producesβ-galactosidase in the presence of N-(3-oxododecanoyl)homoserinelactone. The amount of β-galactosidase can then be compared to astandard to determine if the suspected inhibitor represses the abilityof N-(3-oxododecanoyl)homoserine lactone to stimulate the production ofβ-galactosidase in E. coli MG4.

The language “inhibitors of the autoinducer molecule of P. aeruginosa”is intended to include molecules that interfere with the ability of theautoinducer molecule to stimulate the activity of the LasR protein of P.aeruginosa. Inhibitors include molecules that degrade or bind toN-(3-oxododecanoyl)homoserine lactone. The inhibitors can compete withthe autoinducer molecule not allowing it to perform its intendedfunction.

The language “synergist of the autoinducer molecule of P. aeruginosa” isintended to include molecules that enhance the ability of theautoinducer molecule to stimulate the LasR protein. Synergists includemolecules that bind to either N-(3-oxododecanoyl)homoserine lactone orthe LasR protein.

The present invention also pertains to methods of selecting inhibitoryand synergistic analogs of the claimed autoinducer. The method comprisesmixing a known amount of the autoinducer molecule with a known amount ofthe suspected inhibitory or synergistic analog, measuring the ability ofthe treated autoinducer molecule to stimulate the activity of a selectedgene then determining whether the suspected inhibitory or synergisticanalog represses or enhances the activity of the autoinducer molecule.Actual inhibitory or synergistic analogs of the autoinducer molecule arethen selected.

The present invention further pertains to methods of inhibiting theinfectivity of P. aeruginosa, methods for treating an immunocompromisedhost infected by P. aeruginosa, e.g., a person afflicted with cysticfibrosis, as well as therapeutic compositions. The methods compriseadministering to an individual a therapeutically effective amount of anagent that is capable of inhibiting the activity of the LasR protein.

The language “inhibiting the infectivity of P. aeruginosa” is intendedto include methods of affecting the ability of P. aeruginosa toinitially infect or further infect an organism. This includes usingagents that prevent the LasR protein from activating the transcriptionof extracellular virulence factors such as exotoxin A and elastolyticproteases by P. aeruginosa.

The language “agent” is intended to include molecules that inhibit theability of the LasR protein to activate transcription of extracellularvirulence factors. Agents include inhibitors ofN-(3-oxododecanoyl)homoserine lactone. Agents also include analogs ofN-(3-oxododecanoyl)homoserine lactone that can directly inhibit the LasRprotein of P. aeruginosa or can compete withN-(3-oxododecanoyl)homoserine lactone. Inhibitory agents can be selectedusing the method described above.

The language “administering a therapeutically effective amount” isintended to include methods of giving or applying an agent to anorganism which allow the agent to perform its intended therapeuticfunction. The therapeutically effective amounts of the agent will varyaccording to factors such as the degree of infection in the individual,the age, sex, and weight of the individual, and the ability of the agentto inhibit the activity of the LasR protein of P. aeruginosa in theindividual. Dosage regima can be adjusted to provide the optimumtherapeutic response. For example, several divided doses can beadministered daily or the dose can be proportionally reduced asindicated by the exigencies of the therapeutic situation. Administeringalso includes contacting the agent with the LasR protein outside of anorganism such as with a culture of bacteria.

The agent can be administered in a convenient manner such as byinjection (subcutaneous, intravenous, etc.), oral administration,inhalation, transdermal application, or rectal administration. Dependingon the route of administration, the agent can be coated with a materialto protect the agent from the action of enzymes, acids and other naturalconditions which may inactivate the agent.

The agent can also be administered parenterally or intraperitoneally.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations may contain a preservative toprevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the agentin the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the agent into a sterile vehicle which contains a basicdispersion medium and the required other ingredients from thoseenumerated above.

The agent can be orally administered, for example, with an inert diluentor an assimilable edible carrier. The agent and other ingredients canalso be enclosed in a hard or soft shell gelatin capsule, compressedinto tablets, or incorporated directly into the individual's diet. Fororal therapeutic administration, the agent can be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 1% by weightof active compound. The percentage of the compositions and preparationscan, of course, be varied and can conveniently be between about 5 toabout 80% of the weight of the unit. The amount of agent in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

The tablets, troches, pills, capsules and the like can also contain thefollowing: a binder such as gum gragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin or a flavoring agent such as peppermint,oil of wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it can contain, in addition to materials of the above type, aliquid carrier. Various other materials can be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules can be coated with shellac, sugar or both. Asyrup or elixir can contain the agent, sucrose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anydosage unit form should be pharmaceutically pure and substantiallynon-toxic in the amounts employed. In addition, the agent can beincorporated into sustained-release preparations and formulations.

The language “pharmaceutically acceptable carrier” is intended toinclude solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the agent, use thereof in thetherapeutic compositions and methods of treatment is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the individual to be treated; each unitcontaining a predetermined quantity of agent is calculated to producethe desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the novel dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the agent and the particular therapeuticeffect to be achieve, and (b) the limitations inherent in the art ofcompounding such an agent for the treatment of P. aeruginosa infectionin individuals.

The principal agent is compounded for convenient and effectiveadministration in effective amounts with a suitable pharmaceuticallyacceptable carrier in an acceptable dosage unit. In the case ofcompositions containing supplementary active ingredients, the dosagesare determined by reference to the usual dose and manner ofadministration of the said ingredients.

The language “an immunocompromised host” is intended to include anorganism that has an immune system that is incapable of reacting topathogens. The host can be immunocompromised due to a genetic disorder,disease or drugs that inhibit immune response. An immunocompromised hostincludes an individual afflicted with cystic fibrosis or who is takingcorticosteroids or immunosuppressive agents.

The language “infected with Pseudomonas aeruginosa” is intended toinclude an organism that is found to have the bacteria, Pseudomonasaeruginosa, present in its body in an infective form. For example,Pseudomonas aeruginosa often infects the lungs of cystic fibrosispatients. Even a small number of Pseudomonas aeruginosa found in anorganism can constitute infection with Pseudomonas aeruginosa.

The present invention further pertains to a culture medium containing asan added compound N-(3-oxododecanoyl)homoserine lactone at aconcentration effective to stimulate or promote cellular metabolism,growth or recovery. For example, the culture medium could be used tosupport growth of Pseudomonas aeruginosa.

The language “culture medium” is intended to include a substance onwhich or in which cells grow. The autoinducer molecule can be includedin commercially available cell culture media. Culture media includebroths, agar, and gelatin.

The present invention also pertains to a method of regulating theexpression of a gene. The method comprises inserting a gene intobacteria chosen for enhancement of gene expression by an agent capableof stimulating the activity of the LasR protein and incubating thebacteria with an agent capable of stimulating the activity of the LasRprotein. The method further can include the steps of allowing the geneexpression to reach a desired level and then incubating the bacteriawith an agent capable of inhibiting the activity of the LasR protein.

The present invention also pertains to a method for detecting thepresence or absence of Pseudomonas aeruginosa in a sample. The methodincludes the steps of obtaining a sample fluid suspected of containingPseudomonas aeruginosa and detecting the presence or absence of theautoinducer molecule as an indication of the presence or absence ofPseudomonas aeruginosa in the sample. The presence or absence of theautoinducer molecule can be detected using the bioassay described below.

The present invention also pertains to analogs of the autoinducermolecule that inhibit the induction of virulence factors by theautoinducer molecule or LasR. The virulence factors include exotoxin A,elastolytic proteases, and an alkaline protease.

The invention is further illustrated by the following non-limitingexamples. The contents of all of the references, published patentapplications, and issued patents cited throughout this application areexpressly incorporated by reference.

EXAMPLES Materials and Methods

Bacterial Strains, Plasmids and Culture Conditions

The E. coli strains used were TBl (Gibco-Bethesda Research LaboratoriesLife Technologies (1984) Focus 6, 4), MG4 (Railing, G., Bodrug, S. andLinn, T. (1985) Mol. Gen. Genet. 201, 379-386), and VJS533 (Stewart, V.J. and Paroles, J. V., Jr. (1988) J. Bacteriol. 170, 1589-1597). The P.aeruginosa strains used were PA01, which contains functional lasR andlasI genes, and PAO-RI, which is a lasR-lasI-mutant derived from PAO1(Gambello, M. J. and Iglewski, B. H. (1991) J. Bacteriol. 173,3000-3009). The plasmids used were pLasI-1, a lasI expression vector(Passador, L., Cook, J. M., Gambello, M. J., Rust, L. and Iglewski, B.H. (1993) Science 260, 1127-1130), pKDT17, which contains a lasB::lacZreporter of lasB promoter activity and lasR under control of the lacpromoter, and pHV200I⁻, which contains the V. fischeri luminescence genecluster with an inactive luxI. These plasmids are all ColEl repliconscontaining an ampicillin-resistance marker. Construction of pKDT17involved cloning into SmaI-digested pUCP18 (Schweizer, H. P. (1991) Gene97, 109-112) an 800-bp lasR fragment from pMJG1.7 (Gambello, M. J. andIglewski, B. H. (1991) J. Bacteriol. 173, 3000-3009), which extendedfrom the EcoRV site 59-bp upstream of the lasR transcriptional start tothe AluI site 22 bp beyond the lasR translational stop codon toconstruct pKDT11. In this plasmid, lasR is under control of the lacpromoter. An intermediate construct containing only two PvuII sites wasmade by subcloning the 800-bp fragment in pUC18 to form pKDT13. Thisintermediate construct was digested with PvuII and the plac-lasRfragment was cloned in TthIII-digested pTS400 (Brumlik, M. J. andStorey, D. G. (1992) Molec. Microbiol. 6, 337-344). The resultingplasmid was called pKDT17. The plasmid, pHV200⁻ was derived from the luxregulon-containing pHV200 (Gray, K. M. and Greenberg, E. P. (1992) J.Bacteriol. 174, 4384-4390) by introducing a frameshift mutation in luxI.This was accomplished by digestion of pHV200 with BglII, filling in thesingle-stranded overhangs with taq polymerase and treating with T4 DNAligase.

For production of PAI, cultures of P. aeruginosa PAO1 or E. coli TB1containing pLasI-1 were grown to the late-logarithmic phase in A medium(Maniatis, T., Fritsch, E. F., and Sambrook, J. (1992) MolecularCloning: A Laboratory Manual, ed. Nolan C. (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.), 2nd Ed) supplemented with 0.4%glucose, 0.05% yeast extract, and 1 mM MgSO₄ with shaking at 37° C.,unless otherwise specified. For subsequent use in autoinducer bioassays,E. coli MG4 containing pKDT17 was grown in supplemented A medium at 30°C. with shaking, and E. coli VJS533 containing pHV200I⁻ was grown in Lbroth (Silhavy, T. J., Berman, M. L. and Enquist, L. W. (1984)Experiments with Gene Fusions (Cold Spring Harbor Lab., Cold SpringHarbor, N.Y.), P. 217) at 30° C. with shaking. For plasmid screening andmaintenance, ampicillin (100 μg/ml) was included in E. coli cultures,and carbenicillin (200 ug/ml) was included in cultures of P. aeruginosa.

Plasmid Purification and Transformation Procedure

Plasmids were purified and manipulated (Maniatis, T., Fritsch, E. F.,and Sambrook, J. (1992) Molecular Cloning: A Laboratory Manual, ed.Nolan C. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), 2ndEd) as described elsewhere. The transformation procedure used wasdescribed by Hanahan (Hanahan, D. (1983) J. Mol. Biol. 166, 557-580).

Autoinducer Bioassays

The quantitative assay that was developed for PAI was based on aprevious report that E. coli MG4 containing a plasmid with lasR and alasB::lacZ transcriptional fusion showed a 25-fold induction ofβ-galactosidase, when grown in a medium in which a recombinant E. colicontaining lasI had been grown previously as compared to a medium inwhich E. coli without lasI had been grown. It has been found that thisPAI activity could be extracted and concentrated in ethyl acetate asdescribed for N-(3-oxohexanoyl)homoserine lactone, the V. fischeriautoinducer or VAI (Eberhard, A., Burlingame, A. L., Eberhard, C.,Kenyon, G. L., Nealson, K. H. and Oppenheimer, N.J. (1981) Biochemistry20, 2444-2449; Nealson, K. H. (1977) Arch. Microbiol. 112, 73-79). Forthe bioassay, overnight cultures of E. coli containing the lasB-promoterreporter, pKDT 17 were diluted in supplemented A medium to an opticaldensity of 0.1 at 660 nm, and stored on ice. Each bioassay consisted of2 ml of the cell suspension plus the test sample. After 5.5 h at 30° C.with shaking, β-galactosidase activity was measured by the CHCl₃-sodiumdodecyl sulfate method described by Miller (Miller, J. A. (1976)Experiments in Molecular Genetics (Cold Spring Harbor Lab., Cold SpringHarbor, N.Y.), pp. 352-355). As described in Example 1, there was alinear dose response to autoinducer in this bioassay. Without additionof autoinducer, β-galactosidase activities were 25±10 Miller units, andwith saturating amounts of autoinducer, α-galactosidase activities were1300±200 Miller units. A unit of P. aeruginosa autoinducer activity isdefined as that amount required to achieve 1/2-saturation in thebioassay.

The quantitative assay for VAI was based on those described elsewhere(Kaplan, H. B. and Greenberg, E. P. (1985) J. Bacteriol. 163, 1210-1214;Bainton, N. J., Bycroft, B. W., Chhabra, S. R., Stead, P., Gledhill, L.,Hill, P. J., Rees, C. E. D., Winson, M. K., Salmond, G. P. C., Stewart,G. S. A. B. and Williams, P. (1992)Gene 116, 87-91; Nealson, K H. (1977)Arch. Microbiol. 112, 73-79). E. coli VJS533 containing pHV200I⁻ wasused to test for V. fischeri autoinducer activity. The plasmid, pHV200I⁻contains all of the V. fischeri genes necessary for autoinducibleluminescence in E. coli, however, the gene encoding autoinducersynthase, luxI, is inactivated such that E. coli containing pHV200I⁻ isnot luminescent without addition of VAI. Overnight cultures of E. colicontaining pHV200I⁻ were diluted to an optical density of 0.01 at 660 nmin an assay medium consisting of 0.05% tryptone, 0.03% glycerol, 100 mMNaCl, 50 mM MgSO₄, and 10 mM potassium phosphate, pH 7. Each bioassayconsisted of 1 ml of the cell suspension plus the test sample. After 3hours at room temperature, luminescence was measured by using a BeckmanLS 1800 Scintillation Counter that was set for single photon counting.Synthetic VAI (Kaplan, N. B., Eberhard, A., Widrig, C. and Greenberg, E.P. (1985) J. Radiolabelled Cmpds. and Pharmaceut. 22, 387-395) was usedto construct a standard curve. A unit of activity is defined as thatamount required to achieve a half-maximal response [equivalent toapproximately 25 nM N-(3-oxohexanoyl)homoserine lactone].

Purification of the P. aeruginosa Autoinducer Produced by E. coliContaining pLasI-1

The procedure for PAI purification was based on that describedpreviously for purification of VAI (Eberhard, A., Burlingame, A. L.,Eberhard, C., Kenyon, G. L., Nealson, K. H. and Oppenheimer, N. J.(1981) Biochemistry 20, 2444-2449). Cells and culture fluid wereseparated by centrifugation (10,000×g for 10 min. at 4° C.). The culturefluid was then passed through a 0.2 μm pore-size filter, and thefiltered material was extracted twice with equal volumes of ethylacetate plus 0.1 ml/liter glacial acetic acid. The combined extractswere pooled, water was removed with magnesium sulfate, and the ethylacetate was removed by rotary evaporation at 40-45° C. The residue wasdissolved in 6 ml of ethyl acetate. The ethyl acetate was removed byrotary evaporation and the residue was then extracted with 5 ml ofethanol. The ethanol solution was was dried by rotary evaporation andthe residue was dissolved in ethyl acetate. The ethyl acetate wasremoved and the residue was extracted in 5 ml of ethanol. This ethanolextract was dried, and dissolved in ethyl acetate. Finally, the samplewas dried and dissolved in 0.2 ml of methanol. This sample was furtherpurified by High Performance Liquid Chromatography (HPLC) with a C18reverse phase column (0.46×25 cm). The P. aeruginosa autoinduceractivity was first eluted as a sharp peak at 73 to 78% methanol in alinear 20 to 100% gradient of methanol and water. Fractions constitutingthis peak were pooled, dried by rotary evaporation and the residuedissolved in ethyl acetate plus acetic acid. The ethyl acetate wasremoved, the residue was dissolved in 0.1 ml of methanol and thissolution was subjected to further purification by HPLC, elutingisocratically with 65% methanol in water. Fractions containingautoinducer activity were dried, dissolved in ethyl acetate plus aceticacid and stored at −20° C.

Chemical Synthesis of P. aeruginosa Autoinducer

Synthesis of PAI, N-(3-oxododecanoyl)-L-homoserine lactone was similarto that described by Eberhard et al (Eberhard, A., Burlingame, A. L.,Eberhard, C., Kenyon, G. L., Nealson, K. H. and Oppenheimer, N. J.(1981) Biochemistry 20, 2444-2449) for synthesis of the V. fischeriautoinducer, N-(3-oxohexanoyl)homoserine lactone. The major differencewas that ethyl 3-oxododecanoate was used instead of ethyl3-oxohexanoate. The ethyl 3-oxododecanoate was prepared from decanoylchloride and the dilithio dianion of monoethyl hydrogen malonate asdescribed previously (Wierenga, W. and Skulnick, H. K. (1979) J. Org.Chem. 44, 310-311). The ethylene glycol ketal of ethyl 3-oxododecanoatewas prepared as described for ethyl 3-oxohexanoate (Eberhard, A.,Burlingame, A. L., Eberhard, C., Kenyon, G. L., Nealson, K. H. andOppenheimer, N. J. (1981) Biochemistry 20, 2444-2449) except thatDowex-50 sulfonic acid cation exchange resin was used in place ofp-toluene sulfonic acid as described by Goswami et al (Goswami, A.,Beale, J. M., Jr., Chapman, R. L., Miller, D. W. and Rosazza, J. P.(1987) J. Natural Prod 50, 49-54). The sodium salt was prepared asdescribed (Eberhard, A., Burlingame, A. L., Eberhard, C., Kenyon, G. L.,Nealson, K. H. and Oppenheimer, N. J. (1981) Biochemistry 20,2444-2449). The sodium 3-oxododecanoate was incubated with equimolaramounts of L-homoserine lactone HCl (Sigma Chemical Co., St. Louis, Mo.)and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (Aldrich, Milwaukee,Wis.), and the resulting ethylene glycol ketal ofN-(3-oxododecanoyl)-L-homoserine lactone was deprotected by acidtreatment (Eberhard, A., Burlingame, A. L., Eberhard, C., Kenyon, G. L.,Nealson, K. H. and Oppenheimer, N. J. (1981) Biochemistry 20, 2444-2449)to yield N-(3-oxododecanoyl)-L-homoserine lactone. This compound waspurified by preparative HPLC using a 20 to 100% methanol gradient asdescribed above. A sharp peak of autoinducer activity eluted at 73 and78% methanol, exactly where the major peak of natural autoinducer wasfound to elute. The fractions containing activity were taken to drynessby rotary evaporation, dissolved in ethyl acetate, and this solution wasstored at −20° C. prior to further analysis.

Spectra

Proton NMR was performed at the University of Iowa College of MedicineNMR Facility on a Varian Unity 500 MHz instrument. Infrared spectroscopywas performed on a Nicolet 205 FTIR. Chemical ionization massspectrometry was performed at the University of Iowa College of MedicineMass Spectrometry Facility on a Nermag RIO-10C instrument with adesorption chemical ionization probe. The reagent gas was ammonia.High-resolution fast atom bombardment was performed at the University ofNebraska—Midwest Center for Mass Spectrometry.

Example 1 Extraction and Purification of PAI from Culture Media

Ethyl acetate extracts of E. coli (pLasI-1) culture medium or P.aeruginosa PAO1 culture medium contained PAI activity in an amountequivalent to that in the culture medium prior to extraction. Bioassayson extracts of media from cultures grown to an equivalent opticaldensity of 0.3 in supplemented A medium indicated the P. aeruginosaculture and the E. coli (pLasI-1) culture produced roughly equivalentamounts of PAI (FIG. 1).

Because it has been reported that P. aeruginosa produces the V. fischeriautoinducer, N-(3-oxohexanoyl)homoserine lactone (Bainton, N. J.,Bycroft, B. W., Chhabra, S. R., Stead, P., Gledhill, L., Hill, P. J.,Rees, C. E. D., Winson, M. K., Salmond, G. P. C., Stewart, G. S. A. B.and Williams, P. (1992) Gene 116, 87-91), this compound was tested foractivity as an inducer for the lasB promoter. Also, a homolog of the VAIwas tested, N-(3-oxooctanoyl)homoserine lactone, which has recently beenreported to serve as the autoinducer in conjugal transfer geneactivation in the Gram-negative bacterium, Agrobacterium tumefaciens, aplant pathogen (Zhang, L., Murphy, P. J., Kerr, A. and Tate, M. (1993)Nature 362,446-448). VAI had no detectable activity when tested in theP. aeruginosa autoinducer bioassay at concentrations as high as 1 uM.This compound gives a maximal response at about 50 nM in the V. fischeriautoinducer bioassay. The A. tumefaciens autoinducer (AAI), however, didshow considerable activity (Table 1). This suggested that PAI wasN-(3-oxooctanoyl)homoserine lactone or a compound related to it. TABLE 1Influence of P. aeruginosa , V. fischeri, and A. tumeficiansAutoinducers on lasB Promoter Activity in E. coli MG4 (pKDT17)β-galactosidase Autoinducer added (Miller units)¹ None 26 +/− 2 50 nMN-(3-oxohexanoyl)homoserine lactone (VAI) 33 +/− 3 500 nMN-(3-oxohexanoyl)homoserine lactone (VAI) 34 +/− 1 50 nMN-(3-oxooctanoyl)homoserine lactone (AAI) 56 +/− 5 500 nMN-(3-oxooctanoyl)homoserine lactone (AAI) 733 +/− 50 1.0 unit P.aeruginosa autoinducer²  735 +/− 110 3.0 units P. aeruginosaautoinducer² 1470 +/− 90 ¹ P. aeruginosa autoinducer assays were performed as described inMaterials and Methods. β-galactosidase activity is a measure oflasB::lacZ promoter activity. Numbers are the average of fourexperiments ± the ranges.²The P. aeruginosa autoinducer was an ethyl acetate extract of theculture medium in which E. coli (pLasI-1) was grown.

When PAI from E. coli containing pLasI-1 or P. aeruginosa PAO1 wassubjected to HPLC, a single major peak of activity was observed (FIG.2). In the case of PAI produced by E. coli (pLasI-1) there was a smallpeak of activity that eluted just after the major peak, and with P.aeruginosa there was a small peak of activity that eluted just prior tothe major peak. N-(3-oxooctanoyl)homoserine eluted well ahead of themajor peak of PAI (FIG. 2). This showed that PAI is notN-(3-oxooctanoyl)homoserine lactone. The V. fischeri autoinducer,N-(3-oxohexanoyl)homoserine lactone elutes ahead of N-(3-oxooctanoyl)homoserine lactone. The chromatographic behavior of the PAI, togetherwith the finding that AAI shows activity in the bioassay for P.aeruginosa autoinducer suggests that PAI is an N-acyl-homoserine lactonewith a hydrophobic side-chain, which is longer than that of AAI.

Because it has been reported that P. aeruginosa produces the V. fischeriautoinducer (Bainton, N. J., Bycroft, B. W., Chhabra, S. R., Stead, P.,Gledhill, L., Hill, P. J., Rees, C. E. D., Winson, M. K., Salmond, G. P.C., Stewart, G. S. A. B. and Williams, P. (1992) Gene 116, 87-91),fractions were tested from HPLC for induction of luminescence by usingthe V. fischeri autoinducer bioassay. In fact, there was a peak ofactivity that eluted at the same location as syntheticN-(3-oxohexanoyl)homoserine lactone. There was also a peak that elutedat the same location as synthetic N-(3-oxooctanoyl)homoserine lactone,which shows some activity with V. fischeri (Eberhard, A., Widrig, C.,MacBath, P. and Schineller (1986) Arch. Microbiol. 146, 35-40), and, ashas been shown, is an inducer of the P. aeruginosa lasB (Table 1).However, only low levels of these compounds were present in extracts ofeither P. aeruginosa or E. coli (pLasI-1) culture media. There was nosignificant VAI activity in the PAI peaks shown in FIG. 2. Apparently,this P. aeruginosa autoinducer cannot cross-react with the V. fischeriLuxR protein to activate the luminescence genes.

Example 2 Analysis of Purified P. aeruginosa Autoinducer

The level of PAI activity in extracts of P. aeruginosa PAO1 medium wassimilar to the level in extracts of E. coli containing pLasI-1 (FIG. 1).The autoinducer produced by E. coli containing pLasI-1 was purifiedbecause P. aeruginosa produces a great variety of extracellularcompounds (Nicas, T. I. and Iglewski, B. H. (1985) Can. J. Microbiol.31, 387-392) that could complicate purification of the autoinducer.Using the purification procedure described in the Materials and Methods,approximately 300 μg of PAI was obtained from 3 L of culture fluid.

Analysis of the purified PAI by proton NMR in D₂O at 25° C. showed aspectrum that was remarkably similar to that for VAI (Eberhard, A.,Burlingame, A. L., Eberhard, C., Kenyon, G. L., Nealson, K. H. andOppenheimer, N. J. (1981) Biochemistry 20, 2444-2449) except that theintegrations of the methyl triplet at 0.85 ppm, and the CH₂ multiplet at1.28 ppm indicated the purified PAI had a longer alkyl chain than doesVAI. Based on the similarity between the proton NMR spectrum of PAI andVAI, and the ratio of the methyl triplet and the methylene multiplet, itseemed likely that the purified compound wasN-(3-oxododecanoyl)homoserine lactone. Chemical ionization massspectrometry showed a strong quasimolecular (M+H)⁺ ion with an m/z of298. This is consistent with the conclusion from the proton NMR analysisthat the compound was N-(3-oxododecanoyl)homoserine lactone. Thechemical composition was confirmed by high-resolution fast atombombardment, which more precisely established the mass of the purifiedcompound. The m/z of the (M+H)⁺ was 298.2018. This corresponded to achemical composition of C₁₆H₂₇NO₄. This is the composition ofN-(3-oxododecanoyl)homoserine lactone.

Example 3 Analysis of Synthetic N-(3-oxododecanoyl)homoserine lactone

As a confirmation of the conclusion that the purified PAI wasN-(3-oxododecanoyl)homoserine lactone, this compound was synthesized.The synthetic compound had chromatographic and spectral propertiesindistinguishable from those of the material purified from culturemedium and was biologically active.

The dose response in a P. aeruginosa autoinducer bioassay indicates thehalf-maximal response occurs at 3 to 5 nM N-(3-oxododecanoyl)L-homoserine lactone (FIG. 3). This is in the range found for the V.fischeri (Kaplan, H. B. and Greenberg, E. P. (1985) J. Bacteriol. 163,1210-1214) and A. tumefaciens (Goswami, A., Beale, J. M., Jr., Chapman,R. L., Miller, D. W. and Rosazza, J. P. (1987) J. Natural Prod. 50,49-54) autoinducer systems where half saturations occur at about 50 nMand 5 nM, respectively.

DISCUSSION

Based on the evidence presented in the above examples, the autoinducer,which serves in conjunction with the LasR protein to activate a numberof P. aeruginosa virulence genes, is N-(3-oxododecanoyl)homoserinelactone. This autoinducer has a longer acyl side chain than relatedautoinducers from other bacteria (FIG. 4). It was reported elsewherethat P. aeruginosa produces the V. fischeri autoinducer,N-(3-oxohexanoyl)homoserine lactone (Bainton, N. J., Bycroft, B. W.,Chhabra, S. R., Stead, P., Gledhill, L., Hill, P. J., Rees, C. E. D.,Winson, M. K., Salmond, G. P. C., Stewart, G. S. A. B. and Williams, P.(1992) Gene 116, 87-91), and it was suggested that VAI was the inducerrequired together with LasR for activation of specific P. aeruginosavirulence genes (Jones, S., Yu, B., Bainton, N. J., Birdsall, M.,Bycroft, B. W., Chhabra, S. R., Cox, A. J. R., Golby, P., Reeves, P. J.,Stephens, S., Winson, M. K., Salmond, G. P. C., Stewart G. S. A. B. andWilliams, P. (1993) EMBO J. 12, 2477-2482). The analysis performed inthe above Examples confirmed that a compound, which serves to induce theV. fischeri lux genes is produced by the lasI gene product, and thiscompound has the behavior of V. fischeri autoinducer in HPLC. However,relatively low levels of this compound were synthesized bylasI-containing P. aeruginosa or E. coli (the concentration of thiscompound was 0.5% of the PAI concentration in extracts of P. aeruginosaPAO1), and neither this compound nor authentic VAI (at concentrations ashigh as 500 nM) showed P. aeruginosa autoinducer activity. In fact, thelasI gene product appears to catalyze the synthesis of a number ofrelated compounds by E. coli or P. aeruginosa, including compounds thatbehaved as did N-(3-oxohexanoyl)homoserine lactone andN-(3-oxooctanoyl)homoserine lactone when subjected to methanol-watergradient HPLC. However, N-(3-oxododecanoyl)homoserine lactone is themost abundantly produced of these related compounds. Based on the datain FIGS. 1 and 3, approximately 120 ng/ml of this autoinducer waspresent in a culture of P. aeruginosa grown in supplemented A medium asdescribed. This is equivalent to 400 nM PAI, and is in about 40-foldexcess of the concentrations required with LasR to fully activate thelasB promoter. In contrast, it is estimated that approximately 0.5 ng/mlof VAI was present in the culture of P. aeruginosa.

AAI shows activity not only as the A. tumefaciens (Zhang, L., Murphy, P.J., Kerr, A. and Tate, M. (1993) Nature 362, 446-448) autoinducer, butalso shows activity together with the LuxR protein in activation of V.fischeri luminescence genes (Eberhard, A., Widrig, C., MacBath, P. andSchineller (1986) Arch. Microbiol. 146, 35-40), and together with theLasR protein in activation of the P. aeruginosa lasB (Table 1). Theconcentration of AAI required for activity as the P. aeruginosaautoinducer was higher than the concentration of PAI required foractivity, and the maximal response was lower (Table 1, FIG. 3). VAI,N-(3-oxohexanoyl)homoserine lactone, did not show any detectableactivity as a P. aeruginosa autoinducer (Table 1). Apparently, the LasRprotein shows specificity with respect to the N-acyl side chain length.This also has been shown for the V. fischeri LuxR protein (Eberhard, A.,Widrig, C., MacBath, P. and Schineller (1986) Arch. Microbiol. 146,35-40) and the A. tumefaciens TraR (Zhang, L., Murphy, P. J., Kerr, A.and Tate, M. (1993) Nature 362, 446-448).

It has recently become apparent that regulatory circuits homologous tothe LuxR-LuxI regulatory circuit in V. fischeri and the LasR-LasIcircuit in P. aeruginosa are common to a number of diverse Gram-negativebacteria. At this time, four different autoinducer structures (includingPAI as described here) are known (FIG. 4). The luminescence genes inVibrio harveyi are controlled by N-(3-hydroxybutanoyl)homoserine lactoneor N-(β-hydroxybutyryl)homoserine lactone (Cao, J.-G. and Merghen, E. A.(1989) J. Biol. Chem. 264, 21670-21676; Cao, J.-G. and Meighen, E. A.(1993) J. Bacteriol. 175, 3856-3862) but homologs of luxI and luxR inthis organism have not been identified. A number of bacteria have beenreported to produce N-(3-oxohexanoyl)homoserine lactone, VAI (Bainton,N. J., Bycroft, B. W., Chhabra, S. R., Stead, P., Gledhill, L., Hill, P.J., Rees, C. E. D., Winson, M. K., Salmond, G. P. C., Stewart, G. S. A.B. and Williams, P. (1992) Gene 116, 87-91). In one of these bacteria atleast, Erwinia carotovora, the luxI and luxR homologs expI and expR havebeen identified (Pirhonen, M., Flego, D., Heikinheimo, R. and Palva, E.T. (1993) EMBO J. 12, 2467-2476). The expI gene directs the synthesis ofan autoinducer that is required together with the expR product forinduction of extracellular protease in a fashion reminiscent of PAIcontrol of extracellular protease induction in P. aeruginosa. Conjugaltransfer genes in A. tumifaciens are controlled by AAI (Zhang, L.,Murphy, P. J., Kerr, A. and Tate, M. (1993) Nature 362, 446-448)together with a transcriptional activator encoded by traR (Piper, K. R.,von Bodman, S. B. and Farrand, S. K. (1993) Nature 362,448-450). Thegene or genes required for AAI synthesis have not yet been described.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific embodiments described herein. Such equivalents are consideredto be within the scope of this invention and are covered by thefollowing claims.

1. (canceled)
 2. An autoinducer molecule comprising a molecule of theformula:

wherein n is 2 or 3; Y is O, S, or NH; X is O, S, or NH; and R is aC₁₁-C₁₄ acyl moiety that may be substituted or a moiety having at leastseven members containing a ring structure that may be substituted; themolecule being able to regulate the activity of the LasR protein ofPseudomonas aeruginosa. 3-4. (canceled)
 5. The autoinducer molecule ofclaims 2 wherein R is a C₁₂ acyl moiety. 6-7. (canceled)
 8. Theautoinducer molecule of claim 2 wherein R contains a heterocyclic ringstructure.
 9. The autoinducer molecule of claim 8 wherein theheterocyclic ring structure has five to seven ring members.
 10. Theautoinducer molecule of claim 9 wherein the heterocyclic ring structurecontains oxygen.
 11. The autoinducer molecule of claim 2 wherein Rcontains a carbocyclic ring structure.
 12. The autoinducer of claim 11wherein the carbocyclic ring structure is a fused ring system.
 13. Theautoinducer molecule of claim 2 wherein the molecule is purified fromthe native source.
 14. The autoinducer molecule of claim 13 wherein thenative source is the culture media of Pseudomonas aeruginosa.
 15. Theautoinducer molecule of claim 2 wherein the molecule is synthesized bychemical means.
 16. The autoinducer molecule of claim 2 wherein themolecule is an optically active isomer.
 17. The autoinducer molecule ofclaim 16 wherein the isomer is the L-isomer.
 18. The autoinducermolecule of claim 16 wherein the isomer is the D-isomer. 19-20.(canceled)
 21. An analog of an autoinducer molecule comprising amolecule of the formula

wherein n is 2 or 3; Y is O, S, or NH; X is O, S, or NH; and R is aC₁₁-C₁₄ acyl moiety that may be substituted or a moiety having at leastseven members containing a ring structure that may be substituted; thataffects the activity of the LasR protein.
 22. The analog of claim 21wherein the analog inhibits the autoinducer activity of theN-(3-oxododecanoyl)homoserine lactone.
 23. The analog of claim 21wherein the analog synergistically enhances the autoinducer activity ofN-(3-oxododecanoyl)homoserine lactone.
 24. The analog of claim 21,wherein the analog is an agonist of the LasR protein of Pseudomonasaeruginosa.
 25. The analog of claim 21, wherein the analog is anantagonist of the LasR protein of Pseudomonas aeruginosa. 26-43.(canceled)
 44. A composition comprising an autoinducer molecule of theformula:

wherein n is 2 or 3; Y is O, S, or NH; X is O, S, or NH; and R is aC₁₁-C₁₄ acyl moiety that may be substituted or a moiety having at leastseven members containing a ring structure that may be substituted; themolecule being able to regulate the activity of the LasR protein ofPseudomonas aeruginosa; and a pharmaceutically acceptable carrier.